T cells recognize allogeneic major histocompatibility (MHC) antigens via two distinct pathways of antigen presentation. The direct pathway involves the recognition of intact allogeneic MHC molecules on the surface of donor cells by host T cells, whereas the indirect pathway involves the recognition of donor MHC-derived peptides that are processed by host antigen presenting cells (APCs) and presented as peptides to T cells. CD4 T cells that recognize allogeneic antigens through the indirect pathway play a major role in allograft rejection, yet the requirements for activating CD4 T cells through the indirect pathway remain undefined. We observed that in CD8 T cell depleted and thymectomized mice, CD4 T cell mediated rejection of MHC class I disparate skin allografts was associated with expression of interleukin-4 (IL-4). In vivo, a deficiency in IL-4 production inhibited the activation of alloreactive IL-2-, IL-4-, and IFN-g-producing CD4 T cells in mice challenged with allogeneic skin grafts, resulting in prolongation of skin graft survival. Inhibition of IL-4 during mixed allogeneic lymphocyte cultures prevented the up-regulation of the co-stimulatory molecules CD80 and CD86 on APCs, and resulted in a defect in the ability of APCs to generate sufficient signals for activation of alloreactive T cells. Thus, production of IL-4 is critical for the activation and expansion of CD4 T cells that recognize alloantigen through the indirect pathway. The specific aims are to: 1) Determine the source of IL-4 production during the response to allografts in vivo; 2) Determine the conditions in which IL-4 is required for CD4 T cell activation; 3) Determine how IL-4 affects APC maturation, function and the ability to stimulate alloreactive CD4 effector T cells; and 4) Determine the stage at which IL-4 neutralization prevents acquisition of CD4 T cell effector function. The studies described in this proposal will advance our understanding of mechanisms leading to alloreactive T cell activation and advance our understanding of fundamental aspects of T cell biology. In addition the proposed studies may lead to significant advances in clinical transplantation.